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J. Biochem, 1984, Vol. 96, No. 4 1089-1101
© 1984 Japanese Biochemical Society

research-article

Characterization of Lysosomal Acid Lipase Purified from Rabbit Liver1

Tsuneo IMANAKA, Kimiko AMANUMA-MUTO, Shoji OHKUMA and Tatsuya TAKANO

Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University Sagamiko, Kanagawa 199–01

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25, 000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42, 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40, 000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15–5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 µmol/min/mg protein, and Km values of 50, 70, 200, and 40 µM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains > long chains >> short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.

1This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. A preliminary report of parts of this work has been presented in FEBS Lett. (15).


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 J. Biol. Chem.Home page
S. Sheriff, H. Du, and G. A. Grabowski
Characterization of Lysosomal Acid Lipase by Site-directed Mutagenesis and Heterologous Expression
J. Biol. Chem., November 17, 1995; 270(46): 27766 - 27772.
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